American visceral leishmaniasis is a zoonotic disease caused by Leishmania infantum (= L. chagasi) and transmitted between dogs and humans by sand flies. Thousands of cases of visceral leishmaniasis (VL) are reported annually and mortality ranges between 7 and 10%. VL control programs are inefficient principally due to the low sensitivity of diagnostic methods to identify infected dogs, which are the urban reservoir hosts of VL. Success of VL control programs hinges on the availability of a sensitive, inexpensive and field-applicable test. This would allow control programs to intervene effectively on infected dogs by culling, treating or protecting them against sand fly bites. In this project we propose to evaluate a sensitive field-applicable molecular test that we developed in our laboratory with the goal of providing a novel diagnostic tool for VL control programs. Our Central Hypothesis is that a novel point-of-care Recombinase Polymerase Amplification test (RPA) test will be more sensitive than the currently used rK39 serological test to identify dogs at early stages of L. infantum infection before they progress to the infectious stage. Our RPA test coupled with a dual labeled probe and lateral flow strip (RPA-LF) allows reading the result with the naked eye in < 1 hour. The test has a sensitivity equivalent to quantitative-PCR (0.1 parasites per reaction which is equivalent to 40 parasites/mL blood). Preliminary results in a group of clinically normal dogs from an endemic area showed that RPA-LF was more sensitive than rK39 to detect subclinical infections (51.9% vs. 14.3% positivity, respectively; p= 0.01). The test also detected L. infantum in non-invasive mucosal samples. Aim 1 will determine the efficacy of RPA-LF vs. rK39 to identify dogs at early stages of infection before they represent an epidemiological risk. Approach: we will screen asymptomatic dogs from endemic cities in Brazil and Argentina using RPA-LF (blood, ear skin and mucosal cells) and rK39 serological test. Statistical analysis will determine the sensitivity of rK39 and RPA-LF, having qPCR as gold standard. Agreement between RPA-LF and rK39 will be evaluated. Aim 2 will determine disease progression and time to vector infectivity of dogs found positive by RPA-LF vs. rK39. Approach: A cohort of dogs positive by rK39 or RPA-LF will be followed clinically and parasitologically by xenodiagnosis (feeding of colonized sand flies on dogs) for 12 months. The differences between the two groups (positive rK39 or RPA-LF) in rate of disease progression and time to infectivity to sand flies will be evaluated. At the completion of the study we anticipate having a validated, low cost, point-of-care field test that will enable timely control interventions to reduce urban transmission of VL to humans.